Thesis defense of Marc Moron

The influence of various thermodynamic parameters on protein-protein interaction, as well as the phase behavior of concentrated protein solutions, is of fundamental importance for many physiological processes. This work focuses in particular on the influence of hydrostatic pressure and temperature on the kinetics and dynamics of liquid-liquid phase separation (LLPS) in lysozyme solutions. On the one hand, LLPS performs functional tasks, such as the formation of membrane-less organelles or the transmission of cellular signals. On the other hand, a misregulated LLPS is involved in pathogenic processes, such as the development of Alzheimer’s disease or cataract. Therefore, it is of great importance to fully understand the underlying mechanisms of LLPS. In the past, many studies dealt with the investigation of protein-protein interaction and phase behavior in concentrated protein solutions by small-angle X-ray scattering. However, in these studies, the system was characterized after the LLPS was complete, resulting in the loss of information about the kinetics and dynamics during the formation of the condensed phase. Therefore, in this work, X-ray photon correlation spectroscopy (XPCS) measurements were performed on concentrated protein solutions during LLPS. XPCS is the X-ray analogue of dynamic light scattering (DLS), with the difference that coherent X-ray radiation is used instead of laser light. Due to the smaller wavelength of the radiation, the dynamics can be studied on shorter length scales, even in turbid samples. The focus of this work was to study the kinetics and dynamics as a function of quench depth. In the case of hydrostatic pressure, it was shown that as the quench depth increases, the growth of the concentrated phase slows down and the system forms a nanostructured gel network for the largest quench depths. The autocorrelation functions showed two decays, where the fast decay could be attributed to surface formation and the slow decay to the growth process. For the temperature-induced LLPS, much slower dynamics and the formation of larger structures were observed. The second part of this work addressed the study of dynamics in homogeneous protein solutions. By combining DLS and XPCS measurements, the existence of clusters in lysozyme solutions was demonstrated and it was shown that the characteristic times of individual proteins in homogeneous solutions are faster than the repetition rate of the detectors used.

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